Embedding the future of Regenerative Medicine into the open epigenomic landscape of pluripotent human embryonic stem cells

Embed Size (px)

Citation preview

  • 8/18/2019 Embedding the future of Regenerative Medicine into the open epigenomic landscape of pluripotent human embry…

    1/29

    Embedding the Future of Regenerative Medicine into the Open

    Epigenomic Landscape of Pluripotent Human Embryonic Stem

    Cells

    Xuejun H. Parsons1,2,*

    1San Diego Regenerative Medicine Institute, San Diego, CA 92109, USA

    2Xc elthera, Sa n Diego, CA 92109, USA

    Abstract

    It has been recognized that pluripotent human embryonic stem cells (hESCs) must be transformed

    into fate-restricted derivatives before use for cell therapy. Realizing the therapeutic potential of pluripotent hESC derivatives demands a better understanding of how a pluripotent cell becomes

    progressively constrained in its fate options to the lineages of tissue or organ in need of repair.

    Discerning the intrinsic plasticity and regenerative potential of human stem cell populations reside

    in chromatin modifications that shape the respective epigenomes of their derivation routes. The

    broad potential of pluripotent hESCs is defined by an epigenome constituted of open conformation

    of chromatin mediated by a pattern of Oct-4 global distribution that corresponds genome-wide

    closely with those of active chroma tin modifications. Dynamic alterations in chromatin states

    correlate with loss-of-Oct4-associated hESC differentiation. The epigenomic transition from

    pluripotence to restriction in lineage choices is characterized by genome-wide increases in histone

    H3K9 methylation that mediates global chromatin-silencing and somatic identity. Human stem

    cell derivatives retain more open epigenomic landscape, therefore, more developmental potentialfor scale-up regeneration, when derived from the hESCs in vitro than from the CNS tissue in

    vivo. Recent technology breakthrough enables direct conversion of pluripotent hESCs by small

    molecule induction into a large supply of lineage-specific neuronal cells or heart muscle cells with

    adequate capacity to regenerate neurons and contractile heart muscles for developing safe and

    effective stem cell therapies. Nuclear translocation of NAD-dependent histone deacetylase SIRT1

    and global chromatin silencing lead to hESC cardiac fate determination, while silencing of 

    pluripotence-associated hsa-miR-302 family and drastic up-regulation of neuroectodermal Hox

    miRNA hsa-miR-10 family lead to hESC neural fate determination. These recent studies place

    global chromatin dynamics as central to tracking the normal pluripotence and lineage progres sion

    © 2013 Parsons

    This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/3.0), which permits unrestricted use, distribution, and reproductionin any medium, provided the original work is properlycited.*Corresponding author: [email protected].

    Author's contribution

    Author XHP designed, performed, and analyzed the data presented in the manuscript.

    Author XHP wrote and approved the final manuscript.

    COMPETING INTERESTS

    The author declares competing interests. XHP is the founder of Xcelthera and has intellectual properties related to hESCs.

    NIH Public AccessAuthor Manuscript Annu Res Rev Biol. Author manuscript; available in PMC 2014 October 09.

    Published in final edited form as:

     Annu Res Rev Biol. 2013 ; 3(4): 323–349.

    N I  H -P A A u

    t  h or Manus c r i  pt  

    N I  H -P A A ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or M

    anus c r i  pt  

    http://creativecommons.org/licenses/by/3.0http://creativecommons.org/licenses/by/3.0http://creativecommons.org/licenses/by/3.0

  • 8/18/2019 Embedding the future of Regenerative Medicine into the open epigenomic landscape of pluripotent human embry…

    2/29

    of hESCs. Embedding lineage-specific genetic and epigenetic developmental programs into the

    open epigenomic landscape of pluripotent hESCs offers a new repository of human stem cell

    therapy derivatives for the future of regenerative medicine.

    Keywords

    Human embryonic stem cell; stem cell; pluripotent; epigenome; chromatin; regenerative medicine;neurological disease; heart disease; cell therapy

    1. INTRODUCTION

    Human stem cells, both embryonic and somatic, hold great potential for cell replacement

    and regeneration therapies for human diseases. Gene expression analysis has indicated that

    stem cells do not seem to have a common core transcription profile that dictates the

    undifferentiated self-renewing state [1-4]; which suggests that gene expression alone is not

    sufficient to define either plasticity or lineage specification [5-8]. A search for a common set

    of transcribed genes that defines the characters of all stem cell derivatives, known as

    stemness, has been unsuccessful; there is virtually no overlap in the gene expression profilesof various types or derivations of stem cells, in spite of their apparent phenotypic similarity

    [8-13]. There exist overlaps in gene expression between cells of varying lineages yet a lack 

    of overlap in phenotypes that ostensibly seem similar. Even the expression of a lineage-

    defining gene within stem cells seems to require additional epigenetic cues [14,15]. It is

    clear that epigenetic processes are providing additional regulatory dimensions to stem cell

    behavior [5-8].

    The eukaryotic genome is packaged into chromatin, a nucleoprotein complex in which the

    DNA helix is wrapped around an octamer of core histone proteins to form a nucleosomal

    DNA structure, known as nucleosome, that is further folded into higher-order chromatin

    structures with the involvement of other chromosomal proteins [16-19]. Chromatinmodifications, such as DNA methylation and histone modifications, serve as important

    epigenetic marks for active and inactive chromatin states, thus the principal epigenetic

    mechanism in early embryogenesis [20,21]. Regulation of chromatin structure by covalent

    modification of DNA and histones, by ATP-driven chromatin remodeling, and by

    incorporation of alternative histone variants can influence a broad range of cellular

    processes that include transcription, replication, recombination, and DNA repair; therefore,

    chromatin modifications have been implicated in a broad range of developmental processes

    [22,23]. Chromatin modification creates molecular landmarks that establish and maintain

    stage-specific gene expression patterns and global gene silencing during mammalian

    development. The activities of chromatin modification might be targeted to a specific gene

    through a sequence-specific DNA binding factor, which results in a cascade of chromatinregulation events that determine the fine tuning of cellular signaling and ultimately cell-fate

    choices. Therefore, regulation of chromatin-mediated lineage specification has become a

    fundamental mechanism in human stem cell lineage commitment and differentiation.

    However, these processes in human stem cell development, which may involve dynamic

    Parsons Page 2

     Annu Res Rev Biol. Author manuscript; available in PMC 2014 October 09.

    N I  H -P A A 

    ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or 

    Manus c r i  pt  

  • 8/18/2019 Embedding the future of Regenerative Medicine into the open epigenomic landscape of pluripotent human embry…

    3/29

    equilibrium between active and inactive chromatin states and establishment of chromatin

    codes by covalent modific ations on histones and DNA, remain to be understood.

    The growing number of identified stem cell derivatives and escalating concerns for safety

    and efficacy of these cells towards clinical applications have made it increasingly crucial to

    assess the relative risk-benefit ratio of a stem cell to addressing a particular disease [5-8].

    Discerning the intrinsic plasticity and regenerative potential of human stem cell populations

    reside in chromatin modifications that shape the respective epigenomes of their derivation

    routes [5-8]. Chromatin states have been used to characterize and compare the intricate

    plasticity and potential of stem cell populations [5-8]. Derivation of human embryonic stem

    cells (hESCs) provides a powerful in vitro model system to investigate molecular controls in

    human embryogenesis as well as an unlimited source to generate the diversity of human

    somatic cell types for regenerative medicine [24-26]. Pluripotent hESCs have both the

    unconstrained capacity for long-term stable undifferentiated growth in culture and the

    intrinsic potential for differentiation into all somatic cell types in the human body [24-26].

    However, realizing the developmental and therapeutic potential of hESC derivatives has

    been hindered by the inefficiency and instability of generating clinically-relevant functional

    cells from pluripotent cells through conventional uncontrollable and incomplete multi-lineage differentiation [24,25]. Without a practical strategy to convert pluripotent cells direct

    into a specific lineage, previous studies and profiling of hESCs and their differentiating

    multi-lineage aggregates have compromised implications to molecular controls in human

    embryonic development [27-30]. Developing novel strategies for well-controlled efficiently

    directing pluripotent hESCs exclusively and uniformly towards clinically-relevant cell types

    in a lineage-specific manner is not only crucial for unveiling the molecular and cellular cues

    that direct human embryogenesis, but also vital to harnessing the power of hESC biology for

    tissue engineering and cell-based therapies.

    To date, the lack of a clinically-suitable source of engraftable human stem/progenitor cells

    with adequate neurogenic potential has been the major setback in developing safe and

    effective cell-based therapies for regenerating the damaged or lost central nervous system

    (CNS) structure and circuitry in a wide range of neurological disorders. Similarly, the lack 

    of a clinically-suitable human cardiomyocyte source with adequate myocardium

    regenerative potential has been the major setback in regenerating the damaged human heart.

    Given the limited capacity of the CNS and heart for self-repair, transplantation of hESC

    neuronal and heart cell therapy derivatives holds enormous potential in cell replacement

    therapy. There is a large unmet healthcare need to develop hESC-based therapeutic solutions

    to provide optimal regeneration and reconstruction treatment options for normal tissue and

    function restoration in many major health problems. However, realizing the developmental

    and therapeutic potential of hESC derivatives has been hindered by conventional approaches

    for generating functional cells from pluripotent cells through uncontrollable, incomplete,

    and inefficient multi-lineage differentiation [24-30]. Growing evidences indicate that

    incomplete lineage specification of pluripotent cells via multi-lineage differentiation often

    resulted in poor performance of such stem cell derivatives and/or tissue-engineering

    constructs following transplantation [24,25,31]. The development of better differentiation

    strategies that permit to channel the wide differentiation potential of pluripotent hESCs

    Parsons Page 3

     Annu Res Rev Biol. Author manuscript; available in PMC 2014 October 09.

    N I  H -P A A 

    ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or 

    Manus c r i  pt  

  • 8/18/2019 Embedding the future of Regenerative Medicine into the open epigenomic landscape of pluripotent human embry…

    4/29

    efficiently and predictably to desired phenotypes is vital for realizing the therapeutic

    potential of pluripotent hESCs.

    The pluripotent hESC itself cannot be used for therapeutic applications. It has been

    recognized that pluripotent hESCs must be transformed into fate-restricted derivatives

    before use for cell therapy [7]. Realizing the therapeutic potential of pluripotent hESC

    derivatives demands a better understanding of how a pluripotent cell becomes progressively

    constrained in its fate options to the lineages of tissue or organ in need of repair [7]. Recent

    advances and breakthroughs in hESC research have overcome some major obstacles in

    bringing hESC therapy derivatives towards clinical applications, including establishing

    defined culture systems for de novo derivation and maintenance of clinical-grade pluripotent

    hESCs and lineage-specific differentiation of pluripotent hESCs by small molecule

    induction [5-8,25,32-36]. This technology breakthrough enables direct conversion of 

    pluripotent hESCs into a large supply of high purity neuronal cells or heart muscle cells with

    adequate capacity to regenerate CNS neurons and contractile heart muscles for developing

    safe and effective stem cell therapies [5-8,25,32-36]. Transforming pluripotent hESCs into

    fate-restricted therapy derivatives dramatically increases the clinical efficacy of graft-

    dependent repair and safety of hESC-derived cellular products. Such milestone advancesand medical innovations in hESC research allow generation of a large supply of clinical-

    grade hESC therapy derivatives targeting for major health problems. Currently, these hESC

    neuronal and cardiomyocyte therapy derivatives are the only available human cell sources

    with adequate capacity to regenerate neurons and contractile heart muscles, vital for CNS

    and heart repair in the clinical setting.

    The pluripotence of hESCs that display normal stable expansion is associated with a

    globally active acetylated chromatin, as evident by high levels of expression and nuclear

    localization of active chromatin remodeling factors; weak expression or cytoplasmic

    localization of repressive chromatin remodeling factors that are implicated in transcriptional

    silencing; and residual H3 K9 methylation [5,37]. Profiling of chromatin modifications that

    make up the epigenome of pluripotent hESCs indicated that the broad potential of 

    pluripotent hESCs is defined by an epigenome constituted of open conformation of 

    chromatin mediated by a pattern of Oct-4 global distribution that corresponds genome-wide

    closely with those of active chromatin modifications [5,8]. Dynamic alterations in chromatin

    states correlate with loss-of-Oct4-associated hESC differentiation [5]. The epigenomic

    transition from pluripotence to restriction in lineage choices is characterized by genome-

    wide increases in histone H3K9 methylation that mediates global chromatin-silencing and

    somatic identity [5-8]. The intrinsic plasticity and regenerative potential of human stem cell

    derivatives can be differentiated by their epigenomic landscape features, and that human

    stem cell derivatives retain more open epigenomic landscape, therefore, more developmental

    potential for scale-up regeneration, when derived from the hESCs in vitro than from the

    CNS tissue in vivo [7,8]. Having achieved uniformly conversion of pluripotent hESCs to a

    cardiac or neural lineage with small molecule induction, in our recent reports, we further

    profiled chromatin modifications and microRNA (miRNA) expression in order to uncover

    the genetic and epigenetic mechanisms governing early lineage specification direct from the

    pluripotent stage [6-8,34]. Nuclear translocation of NAD (nicotinamide adenine

    Parsons Page 4

     Annu Res Rev Biol. Author manuscript; available in PMC 2014 October 09.

    N I  H -P A A 

    ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or 

    Manus c r i  pt  

  • 8/18/2019 Embedding the future of Regenerative Medicine into the open epigenomic landscape of pluripotent human embry…

    5/29

    dinucleotide)-dependent histone deacetylase SIRT1 and global chromatin silencing lead to

    hESC cardiac fate determination, while silencing of pluripotence-associated hsa-miR-302

    family and drastic up-regulation of neuroectodermal Hox miRNA hsa-miR-10 family lead to

    hESC neural fate determination [6]. These recent studies place global chromatin dynamics

    as central to tracking the normal pluripotence and lineage progression of hESCs. Embedding

    lineage-specific genetic and epigenetic programs into the open epigenomic landscape of 

    pluripotent hESCs offers a new dimension for direct control and modulation of hESCpluripotent fate when deriving clinically-relevant lineages for regenerative therapies.

    2. EPIGENETIC MODIFICATIONS IN STEM CELL DEVELOPMENT

    2.1 Chromatin Modification in the Establishment of Epigenetic Marks

    Lineage-specific differentiation is a complex process that it is better characterized by the

    establishment of epigenetic marks than by specific gene activation. Recent studies indicate

    that epigenetic controls in stem cell fate decisions hold the key to some of the pressing

    questions regarding the underlying mechanisms of their developmental potential [5-8]. The

    development of chromatin/nucleosome-immunoprecipitation-coupled DNA microarray

    analysis (ChIP/NuIP-chip) and chromatin-immunoprecipitation-combined second-generation high-throughput sequencing (ChIP-seq) has provided the technology foundation

    for genome-wide approaches to profile alterations in spatial and temporal patterns of the

    developmental associated epigenetic markers in high-resolution [8,27,28,30,38-40]. Studies

    of chromatin modifications at a genome-wide scale have led to great advances in our

    understanding of the global phenomena of multiple epigenomes of human stem derivatives

    originated from embryos or various tissue types and developmental stages [8]. Large-scale

    profiling of developmental regulators and histone modifications has been used to identify

    epigenetic patterns for defining the phenotypic features of hESCs and their derivatives

    [8,27,28,30,41-42]. Mapping global patterns of chromatin dynamics in human stem cell

    derivatives will identify underlying molecular mechanisms as well as provide reliably

    predictive molecular parameters for comparing their intrinsic plasticity dominating stem cellbehavior prior to transplantation [5-8]. Although genome-wide mapping of histone

    modifications and chromatin-associated proteins have already begun to reveal the

    mechanisms in mouse embryonic stem cell (ESC) differentiation [43], similar studies in

    hESCs are currently lacking due to the difficulty of conventional multi-lineage

    differentiation approaches in obtaining the large number of purified cells, particularly

    neurons and cardiomyocytes, typically required for ChIP-chip and ChIP-seq experiments

    [28,30,40].

    2.2 Histone Methylation

    Chromatin modification includes processes such as DNA methylation and histone

    acetylation, deacetylation, methylation, phosphorylation and ubiquitylation [16-19] (Fig. 1).These processes function cooperatively to establish and maintain active or inactive

    chromatin states in cellular development. Chromatin remodeling enzymes are largely

    involved in the control of cellular differentiation, and loss or gain of function is often

    correlated with pathological events [44]. Modification of core histone tails is far more

    complex than DNA methylation and involves many different histone modification enzymes

    Parsons Page 5

     Annu Res Rev Biol. Author manuscript; available in PMC 2014 October 09.

    N I  H -P A A 

    ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or 

    Manus c r i  pt  

  • 8/18/2019 Embedding the future of Regenerative Medicine into the open epigenomic landscape of pluripotent human embry…

    6/29

    (Fig. 1). Histones are small, highly conserved basic proteins. Local changes of chromatin

    architecture can be achieved by post-translational modifications of histones such as

    methylation, acetylation, phosphorylation, ubiquitination, sumoylation, and ADP-

    ribosylation [19,44,45].

    These epigenomic changes are dynamic and allow for rapid repression or de-repression of 

    specific target genes. In general, acetylation of core histones and methylation of K4 of 

    histone H3 (H3K4me) correlate with transcriptional active (open) chromatin state, whereas

    deacetylation of core histones and methylation of K9 of histone H3 (H3K9me) correlate

    with transcriptional repressed (closed) chromatin state [16-19,46,47]. Previous reports have

    linked histone H3 K27 methylation to the repression of a special set of developmental genes

    in murine and human ESCs and H3 arginine methylation to the pluripotent inner cell mass

    (ICM) development in mouse embryos [27, 48-50]. The bivalent histone methylation marks

    include the H3K4me3 activation and the H3K27me3 repressive modifications confined to

    ESCs [27]. Several histone methyltransferases (HMT), including a histone H3 K4

    methyltransferase and five histone H3 K9 methyltransferases such as SUV39H1, SUV39H2,

    G9a, ESET/SetDB1, and Eu-HMTaseI, have been identified in mammals [21]. Evidences

    indicate that SUV39H functions to methylates histones in heterochromatin, while G9amethylates histones in euchromatin which is essential for early embryogenesis [51,52].

    Histone H3 K9 trimethylation by HMT has been shown as a mark for subsequent DNA

    methylation, suggesting the critical role of chromatin language in cellular development [45].

    Enhancer of Zeste homlog 2 (EZH2), the HMT within Polycomb repressive II complexes, is

    essential for not only methylation of histone H3 on Lys 27 (H3K27me3) but also interaction

    with and recruiting DNA methyltransferases to methylate CpG at certain EZH2 target genes

    to establish firm repressive chromatin structures, contributing to tumor progression and the

    regulation of development and lineage commitment both in ESCs and adult stem cells

    [53-55]. In addition to being involved in Hox gene silencing, the EZH2/Polycomb complex

    and its associated HMT activity are important in biological processes including X-

    inactivation, germline development, stem cell pluripotence, and cancer metastasis [53-55].

    2.3 Histone Acetylation and Deacetylation

    Studies of transcriptional regulation have revealed that many of the transcription

    coactivators, including Gcn5, p300/CBP, PCAF, Tip60, and nuclear hormone receptor

    coactivators such as SRC-1, ACTR, and TIF2, as well as several subunits associated with

    RNA Polymerase II such as TAF(II) 250, TFIIIC and Elp3, contain intrinsic histone

    acetyltransferase (HAT) activity [56]. Acetylation impacts chromatin structure through the

    neutralization of the charge inherent to the amino group of lysine, thereby weakening intra-

    and inter-nucleosomal interactions of the chromatin fiber and facilitating its decondensation

    by increasing accessibility to the nucleosomal DNA [56]. In addition, acetylation is

    recognized or targeted by the bromodomain of a variety of chromatin factors that mediatestranscription activation and ATP-dependent chromatin remodeling through recruitments of 

    other specific regulators [56].

    On the other hand, histone deacetylases (HDAC) are associated with global transcription

    corepressor such as Sin3 and NcoR/SMRT [57-59]. Inhibitors of HDACs have been found

    Parsons Page 6

     Annu Res Rev Biol. Author manuscript; available in PMC 2014 October 09.

    N I  H -P A A 

    ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or 

    Manus c r i  pt  

  • 8/18/2019 Embedding the future of Regenerative Medicine into the open epigenomic landscape of pluripotent human embry…

    7/29

    to cause stem cell differentiation as well as growth arrest, differentiation, and apoptosis of 

    many tumor cells [5,60]. Three classes of HDACs have been identified so far. Class I human

    HDACs include HDAC1, 2, 3, and 8, and are homologous to yeast Rpd3 [17]. Class II

    HDACs, which are expressed in specific tissues, contain a group of large molecules and are

    homologous to yeast Hda1, such as HADC4, 5, 6, 7, and 9 [17]. Class III HDACs consist of 

    a group of NAD-dependent histone deacetylases known as Sirtuin and are homologous to

    yeast Sir2 (silent information regulator 2) that is involved in transcriptional silencing [19].Sir2, the yeast longevity and transcriptional silencing protein, is recruited to DNA by

    chromatin binding factors to spread to the entire locus and mediate gene silencing during

    mating cell type switch [61]. Sir2 in higher organisms plays an essential role in

    heterochromatic silencing and euchromatic repression, and associates with the bHLH

    repressor proteins, the key regulators of development [62, 63]. Analysis of null mutant of 

    Sir2 in mouse suggests that mammalian Sir2 has an essential role in embryogenesis and

    gametogenesis [64]. In vitro reconstitution studies indicate that histone deacetylation by Sir2

    generates a conformational change or rearrangement of histones into a transcriptionally

    repressive chromatin structure [19]. Sir2 human orthologue SIRT1 physically associates

    with DNA cytosine methyltransferase Dnmt1 and can deacetylate acetylated Dnmt1 in vitro

    and in vivo, which has different effects on the functions of Dnmt1 dependent on the lysineresidues [65]. Interestingly, studying of transcriptional silencing in yeast also revealed

    several members of MYST family of HATs such as Sas2 (something about silencing), Sas3,

    and Esa1 (essential Sas2-related acetyltransferase) are also involved in gene silencing [56].

    Human homologues of MYST family of acetyltransferases include the Tip60 (Tat interactive

    protein 60), MOZ (monocytic leukemia zinc finger protein), MORF (MOZ-related factor),

    and HBO1 (histone acetyltransferase bound to ORC). Tip60/p400 complexes have been

    described as regulating mouse ESC gene expression via Nanog and H3K4 methylation [66].

    SIRT1 negatively regulates the activities, functions, and protein levels of hMOF and TIP60

    [67]. That pools of HAT and HDAC are so evolutionally conserved suggests that a

    mechanism similar to the chromatin-mediated cell type switch in yeast may contribute to

    lineage-specification in human stem cell development.

    2.4 Chromatin Remodeling Factors

    Chromatin remodeling factors are ATP-utilizing motor proteins that mediate the interaction

    of proteins with nucleosomal DNA by DNA/nucleosome-translocation [16,68]. ATP-

    dependent nucleosomal remodeling factor hBrm and hBrg1 (components of hSWI/SNF

    chromatin remodeling complex), and hSNF2H (human homolog of ISWI [Imitation Switch],

    a component of hACF/WCRF and hRSF chromatin remodeling complexes) have been

    shown to be involved in cellular functions such as chromatin assembly, chromosome

    structure, global remodeling of nuclei, DNA replication, recombination, and repair [16,69].

    Two murine members of ISWI, SNF2H and SNF2I, display distinct differential expression

    patterns in the brain: SNF2H is prevalent in proliferating cell populations, whereas, SNF2I is

    predominantly expressed in terminally differentiated neurons after birth [70]. Various

    previous reports in flies and mouse show the involvement of chromatin remodeling factors,

    such as Brm (an ATP-dependent chromatin-remodeling factor implicated in mediating

    H3K9 methylation) and REST/NRSF (a HDAC2-associated transcriptional repressor

    complex), in neuronal development and function [71-76]. Chromatin remodeling complexes

    Parsons Page 7

     Annu Res Rev Biol. Author manuscript; available in PMC 2014 October 09.

    N I  H -P A A 

    ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or 

    Manus c r i  pt  

  • 8/18/2019 Embedding the future of Regenerative Medicine into the open epigenomic landscape of pluripotent human embry…

    8/29

    with ubiquitous subunits including two ATPases Brg1 and Brm and HDACs have been

    shown to mediate repression of neuronal-specific genes [72,76-78]. Smyd1/Bop (SET and

    MYND domain containing 1) and members of Class II HDACs (HDAC 5, 7, 9) are involved

    in regulating the development of mouse ventricular cardiomyocytes [79-82]. A muscle-

    specific member of the SWI/SNF complex, BAF60c (BRG1/BRM-associated factor 60 c), is

    essential to activate both skeletal and cardiac muscle programs in mouse [83,84]. Another

    example of a chromatin remodeling factor is Ikaros, a sequence-specific DNA-binding zincfinger protein and an integral component of nucleosomal remodeling and deacetylation

    complexes (NURD) that contains chromatin remodeling factor Mi-2, HDAC1 and HDAC2

    [69,85]. Deregulation of Ikaros has been found to induce leukemia, indicating it is an

    essential regulator of lymphocyte development [22]. Brg1 has been shown to interact with

    the key regulators of pluripotence, Oct4, Sox2, and NANOG, and exhibit a highly correlated

    genome-wide binding patterns with these proteins in mouse ESCs [86,87], suggesting a

    cooperative role of SWI/SNF complexes in keeping the cells in the undifferentiated state

    [69,88]. In addition, chromodomain helicase DNA binding proteins (CHD), which contain

    two chromodomains, hence exhibiting high affinity for methylated histones, especially

    H3K4me2/3, appear to be required for maintaining a open chromatin conformation in mouse

    ESCs [69,89]. However, the precise functions of those chromatin remodeling factors inregulation of hESC pluripotence and different iation remain to be shown.

    2.5 DNA Methylation

    DNA methylation patterns are established and maintained by DNA methyltransferases.

    Methyl-CpG-binding proteins (MBD) are believed to then recruit NURD that contain Mi-2

    (an ATP-dependent chromatin remodeling factor) and HDAC to deacetylate histones and

    induce gene silencing [90-93]. In addition, methylation of histone H3 K9 by HMT can

    trigger the binding of heterochromatin protein 1 (HP1) to methylated histones, which might

    in turn recruit DNA methyltransferases to stabilize the inactive chromatin [94]. Five

    methylated DNA binding proteins (MeCP2, MBD1, MBD2, MBD3, and MBD4) and three

    active DNA cytosine methyltransferases (Dnmt1, Dnmt3a, and Dnmt3b) have been

    identified in mammals. Dmnt1 is a ubiquitously expressed maintenance methyltransferase

    and functions to restore DNA methylation patterns after DNA replication, while Dnmt3a and

    Dnmt3b function to initiate de novo methylation and establish new DNA methylation

    patterns during development [95]. Targeted deletions of Dmnt1 and MBD3 in mice are

    embryonic lethal [95,96], while mutations in MeCP2 result in deficiency in neural

    development and cause mental retardation (Rett syndrome) [97-101]. Mouse adult neural

    stem cells (NSCs) lacking MBD1 have also been shown deficiency in neural development

    [102]. The deficiency of MBD3 leads to hyperacetylation and loss of mouse ESC

    pluripotence [103].

    DNA methylation and demethylation play an important role in ESC development as well assomatic cell reprogramming and imprint erasure [69,104-107]. DNA methylation is essential

    for normal development and has been implicated in many pathologies including cancer

    [107]. Methylation of CpGs establishes dynamic epigenetic marks that undergo extensive

    changes during cellular differentiation, particularly in regulatory regions outside of core

    promoters [107]. Genome-wide mapping of DNA methylation patterns at proximal promoter

    Parsons Page 8

     Annu Res Rev Biol. Author manuscript; available in PMC 2014 October 09.

    N I  H -P A A 

    ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or 

    Manus c r i  pt  

  • 8/18/2019 Embedding the future of Regenerative Medicine into the open epigenomic landscape of pluripotent human embry…

    9/29

    regions in mouse ESCs suggested that most methylated genes are differentiation associated

    and repressed, while the unmethylated gene set includes many housekeeping and

    pluripotence genes [108]. Somatic cell nuclear transfer and transcription-factor-based

    reprogramming have been used to revert adult cells to an embryonic-like state with

    extremely low efficiencies [109-112]. Pluripotence-inducing factors, most of which are

    known oncogenes, have been used to reprogram mouse and human somatic cells to induced

    pluripotent stem cells (iPS cells) [109-112]. DNA hydroxylase Ten-Eleven Translocation(Tet) family of enzymes, which convert 5-methylcytosine (5mC) to 5-

    hydroxymethylcytosine (5hmC) in various embryonic and adult tissues, facilitates mouse

    pluripotent stem cell induction by promoting Oct4 demethylation and reactivation [105].

    The 5hmC enrichment is involved in the demethylation and reactivation of genes and

    regulatory regions that are important for pluripotence [105]. However, factor-based

    reprogramming can leave an epigenetic memory of the tissue of origin that may influence

    efforts at directed differentiation for applications in disease modelling or treatment and is

    even less effective at establishing the ground state of pluripotence than that of somatic

    nuclear transfer [113]. Somatic cell nuclear transfer and factor-based reprogramming are

    incapable of restoring a correctepigenetic pattern of pluripotent ESCs, which accounts for

    abnormal gene expression, accelerated senescence, and immune-rejection followingtransplantation of reprogrammedcells [114-116]. These major drawbacks have severely

    impaired the utility of reprogrammed or deprogrammed or direct differentiated somatic cells

    as viable therapeutic approaches.

    These evidences suggest that a chromatin-mediated mechanism may be central to

    understanding how potential of a stem cell is restricted such that a particular phenotype

    emerges and, hence, central to judging the plasticity and commitment of a human stem cell.

    Although it is evident that chromatin modification plays a crucial role in epigenetic

    programming in human embryogenesis, the molecular mechanism involved is largely

    unknown. It is known that undifferentiated hESCs express a unique group of genes,

    including Oct-4, as well as possess specific enzymatic activities such as alkalinephosphatase and telomerase [26]. However, none of these markers, in isolation, is

    exclusively expressed by undifferentiated hESCs. Rather, their presence as a group is

    associated with the undifferentiated state [26, 117]. Plasticity and the pre-differentiation

    state remain poorly understood at the molecular level. For mouse ESCs, two independent

    regulatory pathways, the cytokine-dependent LIF/gp130/Stat3 pathway and the cytokine-

    independent pathway mediated by the homeoprotein Nanog are required for the maintenance

    of pluripotence and self-renewal [118,119]. Both pathways require the sustained expression

    of Oct-4. However, the molecular regulation mechanism is different in mouse and human.

    Human and mouse ESCs actually express opposite markers and require distinct conditions

    for maintenance and differentiation [25,120,121]. Unlike mouse ESCs, the maintenance of 

    undifferentiated hESCs does not require LIF and the LIF/Stat3 signaling pathway,suggesting that an entirely different regulatory system might be employed in human

    [122,123]. In embryogenesis, only cells in the ICM express Oct-4. Loss of Oct-4 at the

    blastocyst stage causes these cells to differentiate into trophectoderm, while Oct-4

    expression ensures embryonic germ layer assignment and lineage differentiation [117]. The

    restriction of Oct-4 expression in vivo and in vitro appears more likely to result from

    Parsons Page 9

     Annu Res Rev Biol. Author manuscript; available in PMC 2014 October 09.

    N I  H -P A A 

    ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or 

    Manus c r i  pt  

  • 8/18/2019 Embedding the future of Regenerative Medicine into the open epigenomic landscape of pluripotent human embry…

    10/29

    establishment of a general active chromatin state rather than an outcome of specific

    activators [117]. Investigating epigenetic controls in human stem cell plasticity, potency,

    and fate decisions may unravel the critical regulatory dimension and definition regarding

    how hESCs maintain self-renewal and prevent differentiation as well as how to direct

    lineage-specific differentiation of hESCs.

    3. THE PLURIPOTENCE OF HESCS CONFORMS TO A GLOBALLY ACTIVEHIGHLY ACCESSIBLE EPIGENOME OPEN FOR ENDLESS POSSIBILITY IN

    HUMAN DEVELOPMENT

    3.1 The Normality and Positivity of hESC Open Epigenome Distinguish Pluripotent hESCs

    from iPS Cells and Tissue-Resident Stem Cells

    Pluripotent hESCs, derived from the pluripotent ICM or epiblast of the human blastocyst,

    have both the unconstrained capacity for long-term stable undifferentiated growth in culture

    and the intrinsic potential for differentiation into all somatic cell types in the human body,

    holding tremendous potential for restoring human tissue and organ function. The hESCs are

    not only pluripotent, but also incredibly stable and positive, as evident by that only the

    positive active chromatin remodeling factors, but not the negative repressive chromatin

    remodeling factors, can be found in the pluripotent epigenome of hESCs [5-8,37]. The

    normality and positivity of hESC open epigenome also differentiate pluripotent hESCs from

    any other stem cells, such as the pluripotent iPS cells reprogrammed from adult cells and the

    tissue-resident stem cells [5-8]. Although pluripotent, the iPS cells are made from adult

    cells, therefore, iPS cells carry many negative repressive chromatin remodeling factors and

    unerasable genetic imprints of adult cells that pluripotent hESCs do not have

    [104-106,113,114]. The traditional sources of engraftable human stem cells with neural

    potential for transplantation therapies have been multipotent human neural stem cells

    (hNSCs) isolated directly from the human fetal neuroectoderm or CNS [24,25,124-128].

    Despite some beneficial outcomes, CNS-derived hNSCs appeared to exert their therapeuticeffect primarily by their non-neuronal progenies through producing trophic and/or neuro-

    protective molecules to rescue endogenous host neurons, but not related to regeneration

    from the graft [24,126,127]. Compared to hESCs and their neural derivatives, the epigenome

    of tissue-resident CNS-derived hNSCs is more deacetylated, methylated, and compacted as a

    result of global increases in histone H3K9 methylation mediated repressive chromatin

    remodeling, therefore, stem cells derived from tissues have acquired more silenced

    chromatin and are likely resides at a more advanced stage of development with more limited

    developmental potential and declining plasticity with aging for regeneration [7,8]. So far,

    due to these major limitations in their intrinsic plasticity and regenerative potential, cell

    therapies based on CNS-derived hNSCs have not yielded the satisfactory results expected

    for clinical trials to move forward [129]. Therefore, the intrinsic plasticity and regenerativepotential of human stem cell derivatives can be differentiated by their epigenomic landscape

    features, and that human stem cell derivatives retain more open epigenomic landscape,

    therefore, more developmental potential and plasticity for scale-up regeneration, when

    derived from the hESCs in vitro than from the CNS tissue in vivo [7,8].

    Parsons Page 10

     Annu Res Rev Biol. Author manuscript; available in PMC 2014 October 09.

    N I  H -P A A 

    ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or 

    Manus c r i  pt  

  • 8/18/2019 Embedding the future of Regenerative Medicine into the open epigenomic landscape of pluripotent human embry…

    11/29

    3.2 The Pluripotence of hESCs is Enabled by a Globally Acetylated Open Chromatin

    The pluripotence of hESCs that display normal stable expansion is enabled by a globally

    acetylated, decondensed, highly accessible chromatin associated with high levels of 

    expression and nuclear localization of active chromatin remodeling factors that include

    acetylated histone H3 and H4 (acH3 and acH4); the active ATP-dependent chromatin-

    remodeling factor Brg-1 and hSNF2H; HAT p300; and the class I basal transcription

    maintenance HDAC1 [5-8] (Fig. 2). The association of pluripotence of hESCs with a

    globally open chromatin state conforms to highly dynamic active epigenomic remodeling,

    which provides the molecular foundation for the normal stable pluripotence of hESCs [5,8].

    By contrast, those repressive chromatin remodeling factors that are implicated in

    transcriptional silencing, including repressive chromatin-remodeling factor Brm and Mi-2

    involved in histone H3 K9 methylation or nucleosome deacetylation of NURD; HAT PCAF,

    Tip60, Moz, and HBO-1; tissue-specific class II HDAC4, 5, 6, 7; the class III NAD-

    dependent HDAC SIRT1; and the H3 K9 HMT SUV39H1, were either weakly expressed or

    localized to cytoplasm and/or cell surface, indicating that they are mostly inactive in

    maintaining the pluripotent epigenome of hESCs [5] (Fig. 2). Although undifferentiated

    hESCs display the bivalent histone marks that include the H3K4me3 activation and the

    H3K27me3 repressive modifications, only residual nucleosomal H3 K9 methylation, a

    chromatin modification implicated in transcriptional repression during development, was

    observed in the pluripotent epigenome of hESCs [5,8,27,69]. Residual repressive chromatin

    remodeling implicated in chromatin silencing and transcriptional repression might be

    essential for stabilizing the pluripotent state of hESCs with a globally active open

    epigenome at a normal developmental stage [5]. In fact, aberrant H3 K9 methylation at

    embryonic stage has been associated with DNA hypermethylation and cell malignant

    transformation in abnormal pluripotent embryonic carcinoma cells [130,131].

    Genome-wide profiling of chromatin modifications that make up the epigenome of 

    pluripotent hESCs indicated that the broad potential of pluripotent hESCs is defined by an

    epigenome constituted of open conformation of chromatin mediated by a pattern of Oct-4global distribution that corresponds genome-wide closely with those of active chromatin

    modifications, as marked by either acetylated histone H3 or H4 [8]. Profiling of Oct-4

    binding by genome-wide approaches suggests that Oct-4 binding is widespread and

    particularly enriched for upstream and downstream of transcribed regions [8]. A

    considerable amount of evidence suggests that acetylation of histone H3 and H4 has distinct

    functional and temporal patterns [7,8,19,132]. The H3 modifications seem to be connected

    to proper control of gene expression, whereas acetylation of H4 seems to be most important

    in histone deposition and chromatin structure [7,8,19,132]. It appears that the overall pattern

    of deposition peaks of Oct-4 corresponds more closely with that of acetylated H4 than with

    that of acetylated H3 in general [8].

    The wide distribution pattern of Oct-4 coincident with sites of active chromatin modification

    genome-wide suggested that Oct-4 might play an essential role in the interface of chromatin

    and transcription regulation to maintain a pluripotent epigenome enabled by a globally

    active open chromatin [5,8]. A dynamic progression from acetylated to transient

    hyperacetylated to hypoacetylated chromatin states correlates with loss-of-Oct4-associated

    Parsons Page 11

     Annu Res Rev Biol. Author manuscript; available in PMC 2014 October 09.

    N I  H -P A A 

    ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or 

    Manus c r i  pt  

  • 8/18/2019 Embedding the future of Regenerative Medicine into the open epigenomic landscape of pluripotent human embry…

    12/29

    hESC differentiation, further suggesting that Oct-4 might play an essential role in preserving

    the globally active chromatin state in pluripotent hESCs by maintaining a balanced level of 

    histone acetylation and that changes in Oct-4 expression appeared to promote hESC

    differentiation by allowing alterations in chromatin state [5]. RNA interference directed

    against Oct-4 and HDAC inhibit or analysis support this pivotal link between chromatin

    dynamics and hESC differentiation [5] (Fig. 2). The epigenomic transition from pluripotence

    to restriction in lineage choices is characterized by genome-wide increases in histone H3K9methylation that mediates global chromatin-silencing and somatic identity [5-8]. These

    recent studies reveal an epigenetic mechanism for placing global chromatin dynamics as

    central to tracking the normal pluripotence and lineage progression of pluripotent hESCs

    [5-8]. The transitions between distinct chromatin states, from the open acetylated chromatin

    of the pluripotent hESC to the more compact deacetylated and methylated chromatin of the

    differentiated cells or somatic tissue-resident cells, suggest a self-regulated complex

    dynamic determined by a progression of global chromatin remodeling as lineage

    commitment proceeds through the developmental processes [5-8].

    4. EMBEDDING LINEAGE-SPECIFIC GENETIC AND EPIGENETIC

    DEVELOPMENTAL PROGRAMS INTO THE OPEN EPIGENOMIC

    LANDSCAPE OF PLURIPOTENT HESCS

    4.1 Lineage-Specific Differentiation of Pluripotent hESCs by Small Molecule Induction

    Opens the Door to Investigate Molecular Embryogenesis in Human Development

    Understanding the much more complex human embryonic development has been hindered

    by the restriction on human embryonic and fetal materials as well as the limited availability

    of human cell types and tissues for study. In particular, there is a fundamental gap in our

    knowledge regarding the molecular networks and pathways underlying the CNS and the

    heart formation in human embryonic development. The enormous diversity of human

    somatic cell types and the highest order of complexity of human genomes, cells, tissues, andorgans among all the eukaryotes pose a big challenge for characterizing, identifying, and

    validating functional elements in human embryonic development in a comprehensive

    manner. Many of the biological pathways and mechanisms of lower-organism or animal

    model systems do not reflect the complexity of humans and have little implications for the

    prevention and cure of human diseases in the clinical setting. As a result of lacking a readily

    available human embryonic model system, the mainstream of biomedical sciences is

    becoming increasingly detached from its ultimate goal of improving human health.

    Derivation of hESCs provides not only a powerful in vitro model system for understanding

    human embryonic development, but also unique revenue for bringing the vast knowledge

    generated from the mainstream of biomedical sciences to clinical translation. Development

    and utilization of hESC models of human embryonic development will facilitate rapidprogress in identification of molecular and genetic therapeutic targets for the prevention and

    treatment of human diseases. Such hESC research will dramatically increase the overall

    turnover of investments in biomedical sciences to optimal treatment options for a wide range

    of human diseases.

    Parsons Page 12

     Annu Res Rev Biol. Author manuscript; available in PMC 2014 October 09.

    N I  H -P A A 

    ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or 

    Manus c r i  pt  

  • 8/18/2019 Embedding the future of Regenerative Medicine into the open epigenomic landscape of pluripotent human embry…

    13/29

    To overcome some of the major obstacles in basic biology and therapeutic application of 

    hESCs, recent studies have resolved the elements of a defined culture system necessary and

    sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de

    novo derivation of animal-free therapeutically-suitable hESCs and well-controlled efficient

    specification of such pluripotent cells exclusively and uniformly towards a particular lineage

    by small molecule induction [5,25,32]. These recent reports show that pluripotent hESCs

    maintained under the defined culture conditions can be uniformly converted into a specificneural or cardiac lineage by small molecule induction [5-8,25,32-36]. Retinoic acid (RA)

    was identified as sufficient to induce the specification of neuroectoderm direct from the

    pluripotent state of hESCs and trigger a cascade of neuronal lineage-specific progression to

    human neuronal progenitors (hESC-I hNuP) and neurons (hESC-I hNu) of the developing

    CNS in high efficiency, purity, and neuronal lineage specificity by promoting nuclear

    translocation of the neuronal specific transcription factor Nurr-1 [6-8,25,34,35]. Unlike the

    two prototypical neuroepithelial-like Nestin-positive hNSCs derived from CNS in vivo or

    hESC in vitro via conventional multi-lineage differentiation, these in vitro neuroectoderm-

    derived Nurr1-positive hESC-I hNuPs did not express the canonical hNSC markers, but

    yielded neurons efficiently and exclusively, suggesting that they are a more neuronal

    lineage-specific embryonic neuronal progenitor than the prototypical neuroepithelial-likehNSCs [6-8,25,34,35]. Similarly, we found that such defined conditions rendered small

    molecule nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm

    direct from the pluripotent state of hESCs by promoting the expression of the earliest

    cardiac-specific transcription factor Csx/Nkx2.5 and triggering progression to cardiac

    precursors and beating cardiomyocytes with high efficiently [6,25,32,36]. This technology

    breakthrough enables neuronal or cardiac lineage-specific differentiation direct from the

    pluripotent state of hESCs with small molecule induction, providing much-needed in vitro

    model systems for investigating molecular controls in human CNS or heart development in

    embryogenesis as well as a large supply of clinical-grade human neuronal or heart muscle

    cells across the spectrum of developmental stages for tissue engineering and cell therapies. It

    opens the door for further identification of genetic and epigenetic developmental programs

    underlying hESC neuronal or cardiomyocyte specification.

    Large-scale profiling of developmental regulators and histone modifications by genome-

    wide approaches has been used to identify the developmental associated epigenetic markers

    in high-resolution, including in hESCs and their derivatives [8,27,28,30,41-42]. In addition,

    recently advances in human miRNA expression microarrays and ChIP-seq have provided

    powerful genome-wide, high-throughput, and high resolution techniques that lead to great

    advances in our understanding of the global phenomena of human developmental processes

    [6,30,34,40,133,134]. MiRNAs act as the governors of gene expression networks, thereby

    modify complex cellular phenotypes in development or disorders [135-137]. MiRNAs play a

    key role in regulation of ESC identity and cell lineage in mouse and human ESCs [133-136].MiRNA expression profiling using microarrays is a powerful high-throughput tool capable

    of monitoring the regulatory networks of the entire genome and identifying functional

    elements in hESC development [6,34]. ChIP-seq is a most recently developed technique for

    genome-wide profiling of DNA-binding proteins, histone or nucleosome modifications

    using next-generation deep DNA sequencing technology [40,138,139]. ChIP-seq offers

    Parsons Page 13

     Annu Res Rev Biol. Author manuscript; available in PMC 2014 October 09.

    N I  H -P A A 

    ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or 

    Manus c r i  pt  

  • 8/18/2019 Embedding the future of Regenerative Medicine into the open epigenomic landscape of pluripotent human embry…

    14/29

    higher resolution, less noise and greater coverage than its array-based predecessor ChIP-

    chip, and has become an indispensable tool for studying gene regulation and epigenetic

    mechanisms in development [40,138,139]. ChIP-seq provides a means to rapidly determine

    the precise genomic location of transcription factor binding sites and histone modifications

    on a genome-wide scale. However, without a practical strategy to convert pluripotent cells

    direct into a specific lineage, previous studies are limited to profiling of hESCs

    differentiating multi-lineage aggregates, such as embryoid body (EB), that contain mixedcell types of endoderm, mesoderm, and ectoderm cells or a heterogeneous population of EB-

    derived cardiac or cardiovascular cells that contain mixed cell types of cardiomyocytes,

    smooth muscle cells, and endothelial cells [28-30]. Those previous reports have not

    achieved to utilize high-throughput approaches to profile one particular cell type

    differentiated from hESCs, such as cardiomyocytes [28-30]. Their findings have been

    limited to a small group of genes that have been identified previously, and thus, have not

    uncovered any new regulatory pathways unique to humans [28-30]. Due to the difficulty of 

    conventional multi-lineage differentiation approaches in obtaining the large number of 

    purified cells, particularly neurons and cardiomyocytes, typically required for ChIP and

    ChIP-seq experiments, studies to reveal the mechanism in hESC differentiation remain

    lacking [30,40]. Recent technology breakthrough in lineage-specific differentiation of pluripotent hESCs by small molecule direct induction allows generation of homogeneous

    populations of neural or cardiac cells direct from hESCs without going through the multi-

    lineage EB stage [5-8,25,32-36]. This novel small molecule direct induction approach

    renders a cascade of neural or cardiac lineage-specific progression directly from the

    pluripotent state of hESCs, providing much-needed in vitro model systems for investigating

    the genetic and epigenetic programs governing the human embryonic CNS or heart

    formation. Such in vitro hESC model systems enable direct generation of large numbers of 

    high purity hESC neuronal or cardiomyocyte derivatives required for ChIP-seq analysis to

    reveal the mechanisms responsible for regulating the patterns of gene expression in hESC

    neuronal or cardiomyocyte specification. It opens the door for further characterizing,

    identifying, and validating functional elements during human embryonic neurogenesis orcardiogenesis in a comprehensive manner. Further using genome-wide approaches to study

    hESC models of human CNS or heart formation will not only provide missing knowledge

    regarding molecular human embryogenesis, but also lead to more optimal stem-cell-

    mediated therapeutic strategies for the prevention and treatment of CNS or heart diseases.

    4.2 A Predominant Genetic Mechanism via Silencing of Pluripotence-Associated miRNAs

    and Drastic Up-Regulation of Neuroectodermal Hox miRNAs Governs hESC Neural Fate

    Determination

    Having achieved uniformly conversion of pluripotent hESCs to a cardiac or neural lineage

    with small molecule induction, in our recent reports, we further profiled chromatin

    modifications and miRNA expression in order to uncover the genetic and epigenetic

    mechanisms governing hESC lineage specific progression direct from the pluripotent stage

    [6-8,34]. These in vitro neuroectoderm-derived Nurr1-positive hESC-I hNuPs expressed

    high levels of active chromatin modifiers, including acetylated histone H3 and H4, HDAC1,

    Brg-1, and hSNF2H, retaining an embryonic acetylated globally active chromatin state,

    which suggests that they are a more plastic human embryonic neuronal progenitor [7,8].

    Parsons Page 14

     Annu Res Rev Biol. Author manuscript; available in PMC 2014 October 09.

    N I  H -P A A 

    ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or 

    Manus c r i  pt  

  • 8/18/2019 Embedding the future of Regenerative Medicine into the open epigenomic landscape of pluripotent human embry…

    15/29

    Consistent with this observation, several repressive chromatin remodeling factors regulating

    histone H3K9 methylation, including SIRT1, SUV39H1, and Brm, were inactive in hESC-I

    hNuPs [7,8]. To uncover key regulators, genome-scale profiling of miRNA differential

    expression patterns was used to identify novel sets of human development-initiating

    miRNAs upon small-molecule-induced neural and cardiac lineage specification direct from

    the pluripotent stage of hESCs [6]. A unique set of pluripotence-associated miRNAs was

    down-regulated, while novel sets of distinct cardiac-and neural-driving miRNAs were up-regulated upon the induction of hESC lineage specific differentiation [6]. The expression of 

    pluripotence-associated hsa-miR-302 family was silenced and the expression of Hox

    miRNA hsa-miR-10 family that regulates gene expression predominantly in neuroectoderm

    was induced to high levels in these hESC-derived neuronal progenitors hESC-I hNuPs

    [6,34]. Following transplantation, they engrafted widely and yielded well-dispersed and

    well-integrated human neurons at a high prevalence within neurogenic regions of the brain,

    demonstrating their potential for neuron replacement therapy [7,34]. Genome-scale profiling

    of miRNA differential expression patterns during hESC neuronal lineage-specific

    progression further identified novel sets of stage-specific human embryonic neurogenic

    miRNAs, including silencing of the prominent pluripotence-associated hsa-miR-302 family

    and drastic expression increases of Hox hsa-miR-10 and the let-7 miRNAs [6,34]. ThesemiRNA profiling studies suggested that distinct sets of stage-specific human embryonic

    neurogenic miRNAs, many of which were not previously linked to neuronal development

    and function, contribute to the development of neuronal identity in human CNS formation

    [6,34]. The miR-10 genes locate within the Hox clusters of developmental regulators and are

    coexpressed with a set of Hox genes to repress the translation of Hox transcripts [140]. The

    drastic expression increase of hsa-miR-10 upon exposure of hESCs to RA suggested that RA

    might induce the expression of Hox genes and co-expression of Hox miRNA hsa-miR-10 to

    silence pluripotence-associated genes and miRNA hsa-miR-302 to drive a neuroectoderm

    fate switch of pluripotent hESCs [6,34]. The evolutionarily conserved Hox family of 

    homeodomain transcription factors plays fundamental roles in regulating cell fate

    specification to coordinate body patterning during development [141,142]. Coordinationbetween genetic and epigenetic programs regulates cell fate determination in developmental

    processes [142]. Once established, Hox gene expression is maintained in the original pattern

    by Polycomb (PcG) and trithorax (trxG) group proteins that play essential roles in epigenetic

    developmental processes [141-144]. The PcG and trxG group complexes control the

    maintenance of Hox gene expression in appropriate domains by binding to specific regions

    of DNA and directing the posttranslational modification of histones to silence or activate

    gene expression [141-144].

    Developing strategies for complex 3D multi-cellular models of human embryogenesis and

    organogenesis will provide a powerful tool that enables analysis under conditions that are

    tightly regulated and authentically representing the in vivo spatial and temporal patterns[25]. Therefore, as an authentic and reliable alternative to animal models, we combined our

    breakthrough in establishing hESC neuronal lineage-specific differentiation protocol with

    the advancements in 3D culture microenvironments to develop the multi-cellular 3D CNS

    model targeted for rapid and high fidelity safety and efficacy evaluation of therapeutic

    candidates and cell therapy products. Under 3D neuronal subtype specification conditions,

    Parsons Page 15

     Annu Res Rev Biol. Author manuscript; available in PMC 2014 October 09.

    N I  H -P A A 

    ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or 

    Manus c r i  pt  

  • 8/18/2019 Embedding the future of Regenerative Medicine into the open epigenomic landscape of pluripotent human embry…

    16/29

    these hESC-derived neuronal cells further proceeded to express subtype neuronal markers

    associated with ventrally-located neuronal populations, such as dopaminergic neurons and

    motor neurons [34], demonstrating their potential for neuron regeneration in vivo as stem

    cell therapy to be translated to patients in clinical trials. These recent studies suggest that

    these hESC neuronal derivatives have acquired a neuronal lineage-specific identity by

    silencing pluripotence-associated miRNAs and inducing the expression of miRNAs linked

    to regulating human CNS development to high levels, therefore, highly neurogenic in vitroand in vivo [6,7,34]. Novel lineage-specific differentiation approach by small molecule

    induction of pluripotent hESCs not only provides a model system for investigating human

    embryonic neurogenesis, but also dramatically increases the clinical efficacy of graft-

    dependent repair and safety of hESC-derived cellular products [6-8,25,34,35]. Thus, it offers

    a large supply of plastic human cell source with adequate capacity to regenerate the CNS

    neurons for CNS tissue engineering and developing safe and effective stem cell therapy to

    restore the normal nerve tissue and function.

    4.3 A Predominant Epigenetic Mechanism via SIRT1-Mediated Global Chromatin Silencing

    Governs hESC Cardiac Fate Determination

    A group of miRNAs displayed an expression pattern of up-regulation upon hESC cardiac

    induction by NAM, including the clusters of hsa-miR-1268, 574-5p, 92 family, 320 family,

    1975, 1979, 103, and 107 [6]. Several groups identified miRNAs as the governors of gene

    expression in response to myocardial infarction (MI) and during post-MI remodeling of 

    adult hearts [137]. Signature patterns of miRNAs identified that miR-1, 29, 30, 133, 150,

    and 320 were down-regulated, while miR-21, 23a, 125, 195, 199 and 214 were up-regulated

    during pathological cardiac remodeling in the adult hearts of rodents and humans [137].

    Gain-and loss-of-function studies in mice revealed miR-1 and miR-133 as key regulators in

    cardiac development and stress-dependent remodeling, miR-138 in control of cardiac

    patterning, miR-143/145 and miR126 in cardiovascular development and angiogenesis

    [137]. The miR-1 and miR-133 were previously shown to promote mesoderm and muscle

    differentiation from mouse and human ESCs by repressing nonmuscle gene expression

    [135,137]. Recent miRNA profiling of hESC cardiac induction suggested that a novel set of 

    miRNAs, many of which were not previously linked to cardiac development and function,

    contribute to the initiation of cardiac fate switch of pluripotent hESCs [6].

    Although RA-induced hESC neuronal derivatives retain an embryonic acetylated globally

    active chromatin state, NAM induced global histone deacetylation, significant down-

    regulation of the expression of Brg-1 and HDAC1, and nuclear translocation of the class III

    NAD-dependent histone deacetylase SIRT1 [6]. This observation suggests that NAM

    triggers the activation of SIRT1 and NAD-dependent histone deacetylation that lead to

    globalchromatin silencing yet selective activation of a subset of cardiac-specific genes, and

    subsequently cardiac fate determination of pluripotent hESCs [6]. Sir2 and its humanorthologue SIRT1 are members of the sirtuin family and class III NAD-dependent HDAC

    [19]. These enzymes catalyze a unique reaction in which NAD and acetylated histone are

    converted into deacetylated histone, NAM, and a novel metabolite O-acetyl ADP-ribose

    (OAADPr) [19]. NAM acts as a noncompetitive product inhibitor of the forward

    deacetylation reaction of NAD-dependent SIRT1 and is likely regulating SIRT1 activity in

    Parsons Page 16

     Annu Res Rev Biol. Author manuscript; available in PMC 2014 October 09.

    N I  H -P A A 

    ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or 

    Manus c r i  pt  

  • 8/18/2019 Embedding the future of Regenerative Medicine into the open epigenomic landscape of pluripotent human embry…

    17/29

    vivo [19,145,146]. In humans, there are 7 homologues (SIRT1-7) among which SIRT1, 6, 7

    are classified as nuclear sirtuins, and SIRT2 as cytoplasmic sirtuin, whereas SIRT3, 4, 5

    reside in the mitochondria [147]. SIRT1, 2, 3, 5 are NAD-dependent histone/protein

    deacetylases, whereas SIRT4, 6 are primarily mono-ADP-ribosyl transferases and SIRT7

    exhibits phosphoribosyl-transferase with no deacetylase activity in vitro [147]. NAD-

    dependent SIRT1, which has long been considered as the anti-aging target, is a critical

    epigenetic regulator previously implicated in cardiovascular and metabolic diseases as wellas during embryogenesis [64,147-151]. SIRT1 is expressed at high levels in the heart and

    the nervous system during embryogenesis, suggesting that it is a critical epigenetic regulator

    in embryogenesis [64, 148]. The implication of sirtuins as potential pharmacological targets

    has resulted in a firestorm of work on the seven mammalian sirtuins in less than a decade,

    however, the important connection between the histone deacetylase activity of SIRT1 and

    chromatin has been underappreciated. SIRT1 mediates deacetylation of histones, in

    particular histone H4 K16, and the recruitment of the linker histone H1 [132]. SIRT1 also

    promotes histone H3 K9 methylation by its direct recruitment of HMT SUV39H1 and by

    elevating SUV39H1 activity through conformational changes and deacetylation of 

    SUV39H1 in its SET domain, concomitant with heterochromatin formation [132]. SIRT1

    plays an essential role in heterochromatin silencing and euchromatic repression inmammalian development through association with the bHLH repressor proteins and histone

    rearrangement [19,63,132,152]. Of the four lysine residues in the N-terminal tail of H4 (K5,

    8,12, 16), K16 is the specific target of SIRT1 and plays a unique role in regulating

    chromatin structure [8,19,132]. Histone H4 K16 acetylation is important in epigenetic

    regulation as substantiated by its being the only lysine residue among the N-terminal tails of 

    all histones that is targeted by an exclusive category of HATs as well as HDACs, such as the

    MYST family of HATs and the class III NAD-dependent HADCs to mediate silencing of 

    chromatin locus during phenotype switch in human development [8,19,56,132]. Further

    unveiling the neucleoprotein complex regulation in hESC cardiac lineage specific

    progression towards cardiomyocytes mediated by NAD-dependent histone deacetylase

    SIRT1 will provide critical understanding to the molecular mechanism underlying humanembryonic cardiogenesis, thereby aid the development of more effective and safe stem cell-

    based therapeutic approaches in the heart field.

    5. FUTURE PROSPECTIVES

    To date, the lack of a suitable human neuronal or cardiomyocyte source with adequate CNS

    or myocardium regenerative potential has been the major setback for CNS or myocardial

    tissue engineering and for developing safe and effective cell-based therapies. Recent

    technology breakthrough enables direct conversion of pluripotent hESCs into a large supply

    of high purity neuronal cells or heart muscle cells with adequate capacity to regenerate CNS

    neurons and contractile heart muscles for developing safe and effective stem cell therapies

    [5-8,25,32-36]. Such hESC neuronal and cardiomyocyte therapy derivatives provide

    currently the only available human cell sources with adequate capacity to regenerate CNS

    neurons and contractile heart muscles, vital for CNS and heart repair in the clinical setting.

    Lineage-specific differentiation direct from the pluripotent state of hESCs by small molecule

    induction offers much-needed in vitro hESC model systems for investigating molecular

    Parsons Page 17

     Annu Res Rev Biol. Author manuscript; available in PMC 2014 October 09.

    N I  H -P A A 

    ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or 

    Manus c r i  pt  

  • 8/18/2019 Embedding the future of Regenerative Medicine into the open epigenomic landscape of pluripotent human embry…

    18/29

    controls in human embryonic development as well as a large supply of clinical-grade human

    neuronal and cardiomyocyte therapy derivatives for CNS and myocardial tissue engineering

    and cell therapies [5-8,25,32-36]. Studies to profile novel hESC models of human

    embryonic neurogenesis and cardiogenesis using genome-wide approaches have begun to

    reveal genetic and epigenetic programs in hESC neuronal and cardiac lineage specification

    [6,8,34]. Such genome-wide high-resolution mapping will generate comprehensive

    knowledge of developmental regulators and networks underlying hESC neuronal or cardiacspecification for systems biology approaches and network models of human embryogenesis.

    One of the major challenges in developing hESC therapies is to determine the necessary

    molecular and cellular cues that direct efficient and predicable lineage-specific

    differentiation of pluripotent hESCs. The normal human developmental pathways that

    generate cardiomyocytes and most classes of CNS neurons remain poorly understood. As a

    result, directing hESC differentiation along specific pathways in a systematic manner has

    proved difficult. Unveiling genetic and epigenetic programs embedded in hESC lineage

    specification will not only contribute tremendously to our knowledge regarding molecular

    embryogenesis in human development, but also allow direct control and modulation of the

    pluripotent fate of hESCs when deriving an unlimited supply of clinically-relevant lineages

    for regenerative medicine. Embedding lineage-specific genetic and epigeneticdevelopmental programs into the open epigenomic landscape of pluripotent hESCs will offer

    a new repository of human stem cell therapy derivatives for the future of regenerative

    medicine. The outcome of such research programs will potentially shift current research to

    create new scientific paradigms for developmental biology and stem cell research.

    6. CONCLUSION

    The broad potential of pluripotent hESCs is defined by an epigenome constituted of open

    conformation of chromatin. Recent technology breakthrough enables direct conversion of 

    pluripotent hESCs by small molecule induction into a large supply of lineage-specific

    neuronal cells or heart muscle cells with adequate capacity to regenerate neurons andcontractile heart muscles. Nuclear translocation of NAD-dependent histone deacetylase

    SIRT1 and global chromatin silencing lead to hESC cardiac fate determination, while

    silencing of pluripotence-associated hsa-miR-302 family and drastic up-regulation of 

    neuroectodermal Hox miRNA hsa-miR-10 family lead to hESC neural fate determination.

    Embedding lineage-specific genetic and epigenetic programs into the open epigenomic

    landscape of pluripotent hESCs offers a new dimension for direct control and modulation of 

    hESC pluripotent fate when deriving clinically-relevant lineages for regenerative therapies.

    Acknowledgments

    XHP has been supported by National Institute of Health (NIH) grants from National Institute on Aging

    (NIHK01AG024496) and The Eunice Kennedy Shriver National Institute of Child Health and Human Development(NIHR21HD056530).

    ABBREVIATIONS

    5mC 5-methylcytosine

    Parsons Page 18

     Annu Res Rev Biol. Author manuscript; available in PMC 2014 October 09.

    N I  H -P A A 

    ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or 

    Manus c r i  pt  

  • 8/18/2019 Embedding the future of Regenerative Medicine into the open epigenomic landscape of pluripotent human embry…

    19/29

    5hmC 5-droxymethylcytosine

    acH3/4 Acetylated Histone H3/4

    CHD Chromodomain Helicase DNA Binding Protein

    CNS Central Nervous System

    ChIP/NuIP-chip Chromatin/Nucleosome-Immunoprecipitation-Coupled DNA

    Microarray Analysis

    ChIP-seq Chromatin-Immunoprecipitation-Combined Second-Generation High-

    Throughput Sequencing

    Dmnt DNA Cytosine Methyltransferases

    EB Embryoid Body

    Esa1 Essential Sas2-Related Acetyltransferase

    ESC Embryonic Stem Cell

    EZH2 Enhancer of Zeste Homlog 2

    HAT Histone Acetyltransferase

    H3K4/9/27me Methylation of K4/9/27 of Histone H3

    HBO1 Histone Acetyltransferase bound to ORC

    HDAC Histone Deacetylases

    hESC Human Embryonic Stem Cell

    hESC-I hNu Human Neuron Induced From Human Embryonic Stem Cell

    hESC-I hNuP Human Neuronal Progenitor Induced From Human Embryonic Stem

    Cell

    HMT Histone Methyltransferases

    hNSC Human Neural Stem Cell

    HP1 Heterochromatin Protein 1

    ICM Inner Cell Mass

    iPS Cell Induced Pluripotent Stem Cell

    MBD Methyl-CpG-binding protein

    MI Myocardial Infarction

    miRNA microRNA

    MORF MOZ-Related Factor

    MOZ Monocytic Leukemia Zinc Finger Protein

    NAM Nicotinamide

    NAD Nicotinamide Adenine Dinucleotide

    Parsons Page 19

     Annu Res Rev Biol. Author manuscript; available in PMC 2014 October 09.

    N I  H -P A A 

    ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or 

    Manus c r i  pt  

  • 8/18/2019 Embedding the future of Regenerative Medicine into the open epigenomic landscape of pluripotent human embry…

    20/29

    NSC Neural Stem Cell

    NURD Nucleosomal Remodeling and Deacetylation Complex

    OAADPr O-acetyl ADP-ribose

    PcG Polycomb Group Proteins

    RA Retinoic Acid

    Sas Something About Silencing

    Sir2 Silent Information Regulator

    Tet Ten-Eleven Translocation proteins

    Tip60 Tat interactive protein 60

    trxG Trithorax Group Proteins

    REFERENCES

    1. Ramalho-Santos M, Yoon S, Matsuzaki Y, Mulligan RC, Melton DA. “Stemness”: Transcriptionalprofiling of embryonic and adult stem cells. Science. 2002; 298:597–600. [PubMed: 12228720]

    2. Ivanova NB, Dimos JT, Schaniel C, Hackney JA, Moore KA, Lemischka IR. A stem cell molecular

    signature. Science. 2002; 298:601–4. [PubMed: 12228721]

    3. Sperger JM, Chen X, Draper JS, Antosiewicz JE, Chon CH, Jones SB, et al. Gene expression

    patterns in human embryonic stem cells and human pluripotent germ cell tumors. Proc. Natl. Acad.

    Sci. USA. 2003; 100:13350–5. [PubMed: 14595015]

    4. Richards M, Tan SP, Tan JH, Chan WK, Bongso A. The transcriptome profile of human embryonic

    stem cells as defined by SAGE. Stem Cells. 2004; 22:51–64. [PubMed: 14688391]

    5. Parsons XH. The dynamics of global chromatin remodeling are pivotal for tracking the normal

    pluripotence of human embryonic stem cells. Anatom. Physiol. 2012; S3:002. doi:

    10.4172/2161-0940.S3-002. PMID: 23543848. PMC3609651.

    6. Parsons XH. MicroRNA profiling reveals distinct mechanisms governing cardiac and neural

    lineage-specification of pluripotent human embryonic stem cells. J. Stem Cell Res. Ther. 2012;2:124. doi: 10.4172/2157-7633.1000124. PMID: 23355957. PMC3554249. [PubMed: 23355957]

    7. Parsons XH. An engraftable human embryonic stem cell neuronal lineage-specific derivative retains

    embryonic chromatin plasticity for scale-up CNS regeneration. J. Reg. Med. & Tissue Eng.

    2012; 1:3. doi: 10.7243/2050-1218-1-3. PMID: 23542901. PMC3609668.

    8. Parsons XH. Human stem cell derivatives retain more open epigenomic landscape when derived

    from pluripotent cells than from tissues. J. Regen. Med. 2013; 1:2. doi:

    10.4172/2325-9620.1000103.

    9. Boyer A, Lee TI, Cole MF, Johnstone SE, Levine SS, Zucker JP, et al. Core transcriptional

    regulatory circuitry in human embryonic stem cells. Cell. 2005; 122:947–56. [PubMed: 16153702]

    10. Chen X, Xu H, Yuan P, Fang F, Huss M, Vega VB, et al. Integration of external signaling

    pathways with the core transcriptional network in embryonic stem cells. Cell. 2008; 133:1106–17.

    [PubMed: 18555785]

    11. Efroni S, Duttagupta R, Cheng J, Dehghani H, Hoeppner DJ, Dash C, et al. Global transcription inpluripotent embryonic stem cells. Cell Stem Cell. 2008; 2:437–47. [PubMed: 18462694]

    12. Kim J, Chu J, Shen X, Wang J, Orkin SH. An extended transcriptional network for pluripotence of 

    embryonic stem cells. Cell. 2008; 132:1049–61. [PubMed: 18358816]

    13. Muller FJ, Laurent LC, Kostka D, Ulitsky I, Williams R, Lu C, et al. Regulatory networks define

    phenotypic classes of human stem cell lines. Nature. 2008; 455:401–5. [PubMed: 18724358]

    Parsons Page 20

     Annu Res Rev Biol. Author manuscript; available in PMC 2014 October 09.

    N I  H -P A A 

    ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or 

    Manus c r i  pt  

  • 8/18/2019 Embedding the future of Regenerative Medicine into the open epigenomic landscape of pluripotent human embry…

    21/29

    14. Parker MA, Anderson JK, Corliss DA, Abraria VE, Sidman RL, Park KI, et al. Expression profile

    of an operationally-defined neural stem cell clone. Exp. Neurol. 2005; 194:320–32. [PubMed:

    15992799]

    15. Spivakov M, Fisher AG. Epigenetic signatures of stem-cell identity. Nat. Rev. Genetics. 2007;

    8:263–71. [PubMed: 17363975]

    16. Fyodorov DV, Kadonaga JT. The many faces of chromatin remodeling: SWItching beyond

    transcription. Cell. 2001; 106:523–5. [PubMed: 11551498]

    17. Huang X, Kadonaga JT. Biochemical analysis of transcriptional repression by Drosophila histonedeacetylase 1. J. Biol. Chem. 2001; 276:12497–500. [PubMed: 11278256]

    18. Horn PJ, Peterson CL. Chromatin higher order folding--wrapping up transcription. Science. 2002;

    297:1824–7. [PubMed: 12228709]

    19. Parsons XH, Garcia SN, Pillus L, Kadonaga JT. Histone deacetylation by Sir2 generates a

    transcriptionally repressed nucleoprotein complex. Proc. Natl. Acad. Sci. USA. 2003; 100:1609–

    14. [PubMed: 12571358]

    20. Reik W, Dean W, Walter J. Epigenetic reprogramming in mammalian development. Science. 2001;

    293:1089–93. [PubMed: 11498579]

    21. Li E. Chromatin modification and epigenetic reprogramming in mammalian development. Nat.

    Rev. Genet. 2002; 3:662–73. [PubMed: 12209141]

    22. Georgopoulos K. Haematopoietic cell-fate decisions, chromatin regulation and Ikaros. Nature Rev.

    Immunol. 2002; 2:162–74. [PubMed: 11913067]

    23. Raisner RM, Hartley PD, Meneghini MD, Bao MZ, Liu CL, Schreiber SL, et al. Histone variantH2A.Z marks the 5’ ends of both active and inactive genes in euchromatin Cell. 2005; 123:233–

    48.

    24. Parsons XH, Teng YD, Snyder EY. Important precautions when deriving patient-specific neural

    elements from pluripotent cells. Cytotherapy. 2009; 11:815–24. PMID: 19903095. PMC3449142.

    [PubMed: 19903095]

    25. Parsons XH, Teng YD, Moore DA, Snyder EY. Patents on technologies of human tissue and organ

    regeneration from pluripotent hESCs. Recent Patents on Regenerative Medicine. 2011; 1:142–63.

    PMID: 2335596. PMC3554241. [PubMed: 23355961]

    26. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM.

    Embryonic stem cell lines derived from human blastocysts. Science. 1998; 282:1145–7. [PubMed:

    9804556]

    27. Bernstein BE, Mikkelsen TS, Xie X, Kamal M, Huebert DJ, Cuff J, et al. A bivalent chromatin

    structure marks key developmental genes in embryonic stem cells. Cell. 2006; 125:315–26.[PubMed: 16630819]

    28. Mikkelsen TS, Ku M, Jaffe DB, Issac B, Lieberman E, Giannoukos G, et al. Genome-wide maps of 

    chromatin state in pluripotent and lineage-committed cells. Nature. 2007; 448:553–60. [PubMed:

    17603471]

    29. Cao F, Wagner RA, Wilson KD, Xie X, Fu JD, Drukker M, et al. Transcriptional and functional

    profiling of human embryonic stem cell-derived cardiomyocytes. PLoS One. 2008; 3:e3474.

    [PubMed: 18941512]

    30. Paige SL, Thomas S, Stoick-Cooper CL, Wang H, Maves L, Sandstrom R, et al. A temporal

    chromatin signature in human embryonic stem cells identifies regulators of cardiac development.

    Cell. 2012; 151:221–32. [PubMed: 22981225]

    31. Kriks S, Shim JW, Piao J, Ganat YM, Wakeman DR, Xie Z, et al. Dopamine neurons derived from

    human ES cells efficiently engraft in animal models of Parkinson's disease. Nature. 2011;

    480:547–51. [PubMed: 22056989]

    32. Parsons JF, Smotrich DB, Gonzalez R, Snyder EY, Moore DA, Parsons XH. Defining conditions

    for sustaining epiblast pluripotence enables direct induction of clinically-suitable human

    myocardial grafts from biologics-free hESCs. J. Clinic. Exp. Cardiology. 2012; S9:001. doi:

    10.4172/2155-9880.S9-001. PMID: 22905333. PMC3419496.

    33. Parsons XH. Editorial: Mending the broken heart - Towards clinical application of hESC therapy

    derivatives. J. Clinic. Exp. Cardiology. 2012; 3:e116. doi: 10.4172/2155-9880.1000e116.

    Parsons Page 21

     Annu Res Rev Biol. Author manuscript; available in PMC 2014 October 09.

    N I  H -P A A 

    ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or Manus c r i  pt  

    N I  H -P A A ut  h or 

    Manus c r i  pt  

  • 8/18/2019 Embedding the future of Regenerative Medicine into the open epigenomic landscape of pluripotent human embry…

    22/29

    34. Parsons XH, Parsons JF, Moore DA. Genome-scale mapping of microRNA signatures in human

    embryonic stem cell neurogenesis. Mol. Med. Ther. 2013; 1:2. doi: 10.4172/2324-8769.1000105.

    PMID: 23543894. PMC3609664.

    35. Parsons XH, Teng YD, Parsons JF, Snyder EY, Smotrich DB, Moore DA. Efficient derivation of 

    human neuronal progenitors and neurons from pluripotent human embryonic stem cells with small

    molecule induction. J. Vis. Exp. 2011; 56:e3273. doi: 10.3791/3273. PMID: 22064669.

    PMC3227216. [PubMed: 22064669]

    36. Parsons XH, Teng YD, Parsons JF, Snyder EY, Smotrich DB, Moore DA. Efficient derivation of 

    human cardiac precursors and cardiomyocytes from pluripotent human embryonic stem cells with

    small molecule induction. J. Vis. Exp. 2011; 57:e3274. doi: 10.3791/3274. PMID: 22083019.

    PMC3308594. [PubMed: 22083019]

    37. Azuara V, Perry P, Sauer S, Spivakov M, Jorgensen HF, John RM, et al. Chromatin signatures of 

    pluripotent cell lines. Nat Cell Biol. 2006; 8:532–8. [PubMed: 16570078]

    38. Kim TH, Barrera LO, Zheng M, Qu C, Singer MA, Richmond TA, et al. A high-resolution map of 

    active promoters in the human genome. Nature. 2005; 436:876–80. [PubMed: 15988478]

    39. Schones DE, Zhao K. Genome-wide approaches to studying chromatin modifications. Nat. Rev.

    Genetics. 2008; 9:179–91. [PubMed: 18250624]

    40. Hitchler MJ, Rice JC. Genome-wide epigenetic analysis of human pluripotent stem cells by ChIP

    and ChIP-Seq. Methods Mol Biol. 2011; 767:253–67. doi: 10.1007/978-1-61779-201-4_19.

    [PubMed: 21822881]

    41. Pan G, Tian S, Nie J, Yang C, Ruotti V, Wei H, et al. Whole-genome analysis of histone H3 lysine

    4 and lysine 27 methylation in human embryonic stem cells. Cell Stem Cell. 2007; 1:299–312.

    [PubMed: 18371364]

    42. Zhao XD, Han X, Chew JL, Liu J, Chiu KP, Choo A, et al. Whole-genome mapping of histone H3

    Lys4 and 27 trimethylations reveals distinct genomic compartments in human embryonic stem

    cells. Cell Stem Cell. 2007; 1:286–98. [PubMed: 18371363]

    43. Wamstad JA, Alexander JM, Truty RM, Shrikumar A, Li F, Eilertson KE, et al. Dynamic and

    coordinated epigenetic regulation of developmental transitions in the cardiac lineage. Cell. 2012;

    151:206–20. [PubMed: 22981692]

    44. Pattaroni C, Jacob C. Histone methylation in the nervous system: functions and dysfunctions. Mol

    Neurobiol. 2013; 47:740–56. [PubMed: 23161382]

    45. Shahbazian MD, Zhang K, Grunstein M. Histone H2B ubiquitylation controls processive

    methylation but not monomethylation by Dot1 and Set1. Mol Cell. 2005; 19:271–7. [PubMed:

    16039595]

    46. Jenuwein T, Allis CD. Translating the histone code. Science. 200